The long term objective of this proposal is to study a novel mRNA splicing pathway involving at least one of the tRNA splicing factors. Recently, genetic analysis from Peter Walter's lab has identified a mutation in the tRNA ligase gene that impairs splicing of Hac1 pre-mRNA in yeast. The Hac1 gene encodes a transcription factor involved in the unfolded protein response (UPR) pathway. Splicing of Hac1 pre-mRNA removes a 252 nt intron and generates a longer, more stable form of protein. This is the first known example of mRNA splicing using elements of the tRNA processing pathway. PI has accumulated an extensive expertise and a battery of reagents needed for analysis of tRNA ligase, the activity implicated in Hac1 splicing. The present proposal will focus on characterization of this novel splicing pathway and the role of tRNA ligase. Specific Aim 1 will focus on the structure of pre-mRNA substrate and requirements for splice site signals. Aim 2 will explore mutations in the ligase gene that are Hac1-specific, tRNA-specific, and those that affect both splicing pathways, to study the relationship between the two splicing processes. The third aim will involve a genetic screen to detect other gene products involved in Hac1 splicing. Finally, in vitro reconstitution system for Hac1 splicing will be established to study the details of tRNA ligase function and the mechanism of the reaction.